The development of the ocular lens involves controlled proliferation and progressive differentiation of equatorial epithelial cells into terminally differentiated lens fibers. Intracellular signaling mechanisms that regulate these cellular processes and their possible role in cataractoqenesis have not been characterized. We hypothesize that Rho GTPases (Rho and Rac) play a critical role in lens growth and development and in maintenance of lens structural integrity by regulating cytoskeletal organization and growth factor stimulated stress-response signaling pathways. Strong evidence for this hypothesis derives from our recent studies based on pharmacological and genetic modulation of Rho GTPase function in different model systems including lens organ and epithelial cell culture, and a C3-exoenzyme transgenic mouse model. Inactivation of Rho GTPase in cell culture and in vivo resulted in alterations of lens epithelial and fiber cell morphology, actin cytoskeletal orqanization and cell adhesive characteristics. Additionally, the Rho GTPase functional knock-out mouse developed cataract and exhibited differential expression of genes encodinq proteins of the extracellular matrix and basement membrane, as well as proteins of stress signaling and apoptotic pathways. Further, several growth factors activated Rho GTPases (both Rho and Rac) in lens epithelial cells. These observations support a critical role for Rho GTPase (Rho and Rac)-mediated signaling mechanisms in lens development, growth and integrity. To elucidate the mechanistic basis (es) by which Rho GTPases regulate lens growth and development, we propose to investigate 1. The roles of RhoA and RhoB GTPases and their down stream effector-Rho kinase, in lens epithelial cell proliferation and cell fate using a RhoB knockout mouse, and by overexpressing dominant neqative mutants of RhoA or Rho kinase in a human lens epithelial cell line using adenoviral vectors. 2. The role of Rac GTPase and reactive oxygen species (ROS)-activated stress signaling pathways (C-jun-N-terminal kinase-JNK1 and p38 MAPK) in growth factor-induced proliferation and cell survival (Akt and NFkappaB) of human lens epithelial cells using an NADPH oxidase inhibitor and by overexpression of dominant negative and constitutively active mutants of Rac GTPase. 3. Regulation of lens protein tyrosine phosphatase activity by Rac/NADPH oxidase in organ-cultured lenses and in lens epithelial cells, in the context of lens growth and epithelial cell proliferation. The completion of these studies should unravel the significance of Rho/Rho kinase and Rac GTPase-mediated signaling and the role of stress -activated signaling pathways in proliferation, survival and apoptosis of lens epithelial cells. Better understanding of signal transduction pathways regulating lens growth and maintenance of lens transparency could provide insights into the etiology of cataract formation and into novel approaches towards developing medical treatments for certain types of cataract.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY012201-09
Application #
7189016
Study Section
Special Emphasis Panel (ZRG1-VISA (01))
Program Officer
Araj, Houmam H
Project Start
1999-03-01
Project End
2009-05-31
Budget Start
2007-03-01
Budget End
2009-05-31
Support Year
9
Fiscal Year
2007
Total Cost
$261,694
Indirect Cost
Name
Duke University
Department
Ophthalmology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Maddala, Rupalatha; Nagendran, Tharkika; de Ridder, Gustaaf G et al. (2013) L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins. PLoS One 8:e64676
Pattabiraman, Padmanabhan P; Lih, Fred B; Tomer, Kenneth B et al. (2012) The role of calcium-independent phospholipase A2? in modulation of aqueous humor drainage and Ca2+ sensitization of trabecular meshwork contraction. Am J Physiol Cell Physiol 302:C979-91
Maddala, Rupalatha; Chauhan, Bharesh K; Walker, Christopher et al. (2011) Rac1 GTPase-deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival. Dev Biol 360:30-43
Maddala, Rupalatha; Skiba, Nikolai P; Lalane 3rd, Robert et al. (2011) Periaxin is required for hexagonal geometry and membrane organization of mature lens fibers. Dev Biol 357:179-90
Pattabiraman, Padmanabhan P; Rao, Ponugoti Vasantha (2010) Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells. Am J Physiol Cell Physiol 298:C749-63
Inoue-Mochita, Miyuki; Inoue, Toshihiro; Epstein, David L et al. (2009) RGS2-deficient mice exhibit decreased intraocular pressure and increased retinal ganglion cell survival. Mol Vis 15:495-504
Rao, P Vasantha; Maddala, Rupalatha (2009) Abundant expression of ponsin, a focal adhesion protein, in lens and downregulation of its expression by impaired cytoskeletal signaling. Invest Ophthalmol Vis Sci 50:1769-77
Rao, P Vasantha; Ho, Tammy; Skiba, Nikolai P et al. (2008) Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins. Biochem Biophys Res Commun 368:508-14
Rao, P Vasantha (2008) The pulling, pushing and fusing of lens fibers: a role for Rho GTPases. Cell Adh Migr 2:170-3
Maddala, Rupalatha; Reneker, Lixing W; Pendurthi, Bhavana et al. (2008) Rho GDP dissociation inhibitor-mediated disruption of Rho GTPase activity impairs lens fiber cell migration, elongation and survival. Dev Biol 315:217-31

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